Molecular phenotypes of osteoarthritis

Abstract

The aim of this study was to analyze the anti-inflammatory effect of VA692, in comparison with celecoxib. In addition, we would establish if the new compound presents any advantages over other COX-2 inhibitors already available in therapeutic use. By iTRAQ methodology, we quantitatively analyzed the different expressed profiles in T/C-28a2 cell line treated with the studied drugs in presence of IL-1b. Methods: Human T/C-28a2 chondrocytes cell line were generated by Goldring group. Human articular cartilage was obtained from femoral heads of five OA patients. Cells were incubated with VA692 and celecoxib (1, 0.5 and 0.2mM) with interleukin (IL)-1b (5ng/ml) for 48hrs. The expression of inflammatory cytokines and anti-oxidant enzymes was evaluated by quantitative qRT-PCR, prostaglandins (PG)-E 2 release by ELISA. Apoptosis levels and ROS production were evaluated by flow cytometry. Proteins extracted by T/C-28a2 cell line was employed to carry out western blot analysis and the iTRAQ methodology. Statistical analysis was performed by ANOVA and Bonferroni multiple comparison tests. Results: IL-1b-stimulated chondrocytes showed a significant increase (p<0.001) of COX-2, IL-1b, IL-6, IL-8, superoxide dismutase (SOD)-2 and catalase (CAT) gene expression, as well as of PGE 2 levels in OA chondrocyte and in T/C-28a2 cell line. The tested drugs significantly counteracted the effect of IL-1b, with a better modulation by VA692 1mM in T/C-28a2 (p<0.01 for COX-2, IL-1b, IL-8, CAT; p<0.001 for IL-6, SOD-2 and PGE 2). Regarding apoptosis and mitochondrial superoxide anion production, the new drug was able to significantly reduce (p<0.05) their increase induced by IL-1b (p<0.05). Proteomic analysis led to the identification of 797 proteins in T/C-28a2 cell line, 123 of which were significantly modulated by VA692 in presence of IL-1b (p<0.001), while 34 were modulated by IL-1b alone (p<0.05). 21 proteins were commonly modulated by the two groups, indicating that 101 proteins were regulated by VA692 in a specific manner. Among the proteins downregulated by VA692, some with structural function were detected, responsible for cytoskeleton reorganization, as well as chaperones (heat shock proteins) and glycolitic enzymes. Proteins involved in calcium metabolism and in ribosome biogenesis resulted up-regulated instead, as well as SOD-2 as confirmed by western blot analysis. Conclusions: Our data demonstrated the anti-inflammatory effect of VA692, suggesting also its anti-apoptotic and anti-oxidant role. The proteomic analysis demonstrated that VA692 induced not only an antiinflammatory regulation in chondrocytes but, interestingly, seemed to regulate their anabolic response. On the basis of our results, we can hypothesize that VA692 could present any advantages over other COX-2 inhibitors already available for therapeutic use. However, further in vitro and in vivo experiments are necessary to elucidate and confirm the importance of this pharmacological compound before its use in the therapeutic approach of joint diseases.

Endothelium-dependent molecular mechanisms of articular cartilage and subchondral bone remodeling in conditions of cardiovascular comorbidity

“…Отложение липидов в тканях, как известно, приводит к активации матриксных металлопротеиназ и деградации межклеточного матрикса. С другой стороны, окисление липидов ведет к NADPH-зависимому окислительному стрессу [4]. Накопление активных форм кислорода в свою очередь запускает эндотелиальную и экстравазальную экспрессию VEGF-A, способствует миграции и дифференцировки CD34-позитивных клеток [15].…”

“…Все больше публикаций свидетельствует в пользу важной роли сердечно-сосудистых факторов в инициации и прогрессировании ОА. В частности, известно, что артериальная гипертония (АГ), гиперлипидемия (ГЛ), системное сосудистое ремоделирование ассоциированы со структурными и клиническими проявлениями ОА [3,4]. В понимание причинно-следственных связей вносят результаты исследований, показавших, что формирование сердечно-сосудистых паттернов предшествует дебюту ОА [5].…”